1. Volume 25, Issue 3, Pages 270-283 (May 2013)
Mitotic Rounding Alters Cell Geometry to Ensure Efficient Bipolar Spindle Formation 
Oscar M. Lancaster, Maël Le Berre, Andrea Dimitracopoulos, Daria Bonazzi, Ewa Zlotek-Zlotkiewicz, Remigio Picone, Thomas Duke, Matthieu Piel, Buzz Baum 
Developmental Cell 
Volume 25, Issue 3, Pages (May 2013)
DOI: /j.devcel
Copyright © 2013 Elsevier Inc. Terms and Conditions

2. Figure 1 A Failure in Cell Rounding Delays Mitotic Progression
(A) Schematic diagram showing the different methods we used to perturb mitotic cell rounding.
(B) Heights of control (n = 8) and Rap1* (n = 7) cells as the cells entered mitosis. Means and SD from the mean are shown.
(C) Times from NEB to anaphase for control (n = 86) and Rap1*-expressing cells (n = 52). Rap1*-expressing cells exhibited a near 2-fold delay in anaphase onset timing (p < 0.001, unpaired t test).
(D) Representative x-y (maximum projection) and x-z (single plane) confocal images of control and Rap1* cells in interphase and at metaphase on unpatterned and patterned substrates.
(E) Maximum length and height of the cells described in (D) (n ≥ 8).
(F) NEB to anaphase timing for Rap1*-expressing cells on large ovals (n = 56) versus small ovals (n = 35), and large ovals (n = 85) versus lines (n = 46). The time to anaphase onset was significantly increased on large ovals (p < 0.01, unpaired t test).
(G) Representative x-y and x-z images of control cells, cells growing under gels of intermediate (5 kPa) and higher (15 kPa) stiffness, and cells at 5 μm confinement.
(H) Diameter of mitotic cells as a function of gel stiffness. Means and SD from the mean are shown (n = 23, 19, 17, 47, 53, 17, 21). The height of the cells was estimated from the cell diameter assuming an ellipsoid cell geometry. The gray line is a fit from a mechanical model (see Experimental Procedures).
(I) NEB-to-anaphase timing for control cells, cells growing under gels of increasing stiffness, and cells confined to a height of 8, 7, 6, or 5 μm (n = 44, 9, 35, 15, 4, 4, 24, 21, 24, 38, 18). At all levels of confinement, except 8 μm (p = 0.89, unpaired t test), the time to anaphase onset was increased (p < 0.01, unpaired t test). Scale bars represent 20 μm.
See also Figure S1.
Developmental Cell  , DOI: ( /j.devcel )
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3. Figure 2 Flat Mitotic Cells Are Defective in Chromosome Capture
(A) Mitotic spindle formation in control and Rap1*-expressing HeLa cells. Representative time-lapse maximum projection images are shown (see also Movie S1). In flattened Rap1*-expressing cells, chromosomes were lost from the nascent spindle (yellow arrows) or became arrayed along the side of the forming spindle (blue arrows).
(B) Mitotic spindle formation in HeLa cells confined to 5 μm in height. Representative time-lapse maximum projection images are shown (see also Movie S2).
(C) Chromosome area as a function of time in control and Rap1* cells (left graph: each line represents data from one cell). Chromosome area was expressed as a fraction of the initial area at NEB (right graph).
(D) Plots of normalized chromosome area against time were fitted to a parametric model, y = 1 − C(1 − exp(−kt)), where C is the total relative decrease in chromosome area and k is the rate of decrease. A single representative data set with the fitted model (black line) is shown for control and Rap1*-expressing cells. Mean R2 values for control (n = 8) and Rap1* (n = 11) data sets are shown in the inserted bar graph. Error bars represent SD from the mean.
(E) Values of C and k for control (n = 8) and Rap1*-expressing (n = 11) cells are plotted as a box-and-whiskers plot. No significant difference was found in the value of C between control and Rap1* cells (p = 0.626, unpaired t test). The value of k was nearly 2-fold smaller in Rap1* cells (p < 0.01, unpaired t test), indicative of a reduced rate of chromosome gathering. Scale bars represent 20 μm.
See also Figure S2.
Developmental Cell  , DOI: ( /j.devcel )
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4. Figure 3
Chromosomal Capture Is Limited by the Reach of Astral Microtubules
(A) Astral microtubules in fixed control and Rap1* cells. Representative confocal images are shown as maximum projections. Scale bars represent 20 μm.
(B) We calculated the probability (p) of a chromosome falling within range of a microtubule, as represented in this schematic diagram (for details see Experimental Procedures).
(C) The first stages of spindle formation in control and Rap1*-expressing HeLa cells are shown as maximum projection images. Scale bars represent 10 μm. The probability of chromosomes being within range of a microtubule at NEB was mapped onto chromosomes as a grayscale image (pink panels). The darkest regions predict chromosome loss (yellow arrows). These probabilities were then considered independently for both centrosomes (gray panels). Chromosomes within range of one centrosome became arrayed around the centrosome and along the side of the forming spindle (blue arrows).
(D) Rap1*-expressing cell in which centrosomes were located on the same side of the nucleus at NEB; many chromosomes were predicted to be lost from the nascent spindle (yellow arrows). Scale bar is 10 μm.
(E) Percentage of chromosomes with a given probability (p) of being within range of a microtubule at NEB for control (n = 10) and Rap1* (n = 8) cells.
(F) Numbers of chromosomes lost from the nascent spindle 10 min after NEB in control (n = 10) and Rap1*-expressing (n = 10) cells. Rap1* cells exhibited a significant increase in the number of lost chromosomes (p < 0.05, unpaired t test).
(G) Percentage of chromosomes within range of microtubules from one or both poles at progressive time points after NEB for control (n = 6) and Rap1*-expressing (n = 6) cells. Graphs in (E) and (G) show mean values and error bars represent SD from the mean.
See also Figure S3.
Developmental Cell  , DOI: ( /j.devcel )
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5. Figure 4 Confinement in Height Leads to Pole Splitting
(A) Images show representative mitotic spindles in control cells, in flattened cells expressing Rap1*, in cells grown under a 5 kPa gel, or under fixed confinement. Scale bar represents 20 μm. At high levels of confinement (5 and 3.5 μm), multipolar spindles (yellow arrows) and spindles with numerous uncongressed chromosomes (blue arrow) were observed.
(B) Time-lapse images of mitotic HeLa cells under 5 μm confinement (see also Movie S3). A magnified view of the splitting pole is shown in the insets and the yellow arrows indicate the new poles formed after splitting. The dashed white lines represent pole-to-chromosome distances measured in (D). Scale bar represents 20 μm.
(C) Defects in spindle architecture were quantified (n = 100, 25, 100, 45, 35, 22, 81, 43, 84, 163, 160, 51, 15).
(D) Data showing the distances from the spindle pole to the edge of the metaphase plate in control and Rap1*-expressing cells, in cells at 8 μm confinement, and in cells at 5 and 3 μm confinement before and after pole splitting (n = 18, 18, 19, 17, 13, 9, 4).
See also Figure S4.
Developmental Cell  , DOI: ( /j.devcel )
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6. Figure 5
Rescuing Mitotic Defects in Flat Cells by Increasing Microtubule Lengths
(A) Representative maximum projection time-lapse images of the first stages of spindle formation in control and MCAK-depleted HeLa cells expressing Rap1*. The probability of chromosomes being within range of a microtubule at NEB was calculated as shown in Figure 3B and mapped onto the chromosomes as a grayscale image (pink panels). The darkest regions predict chromosome loss in control depleted cells (top panel). Distant chromosomes (yellow arrows) were successfully captured in MCAK-depleted cells (bottom panels). Scale bar represents 10 μm.
(B) Representative time-lapse images of control and MCAK-depleted cells growing under 5 μm confinement. Lost chromosomes were observed during early stages of spindle formation (yellow arrow) in control depleted cells but not in cells lacking MCAK. Pole-splitting events (blue arrows) were rescued by MCAK RNAi. Scale bar represents 20 μm.
(C) Plot showing the number of chromosomes lost from the nascent spindle 10 min after NEB in control (n = 24) and MCAK-depleted (n = 20) Rap1* cells. Fewer chromosomes were lost from the nascent spindle in flat Rap1*-expressing cells when microtubules were lengthened in the absence of MCAK (p < 0.05, unpaired t test).
(D) Percentage of monopolar spindles in control and MCAK-depleted cells confined in height to 5 μm (n = 267, 342; in five experiments). Data are represented as means; error bars represent SD between experiments. Fewer multipolar spindles were observed in mitotic cells confined to 5 μm in height when microtubules were lengthened in the absence of MCAK (p < 0.01, unpaired t test).
See also Figure S5.
Developmental Cell  , DOI: ( /j.devcel )
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7. Figure 6
F-Actin Is Only Essential for Mitosis under Conditions of Confinement
(A) Schematic diagram showing changes in actin organization during mitosis.
(B) Time-lapse images of mitosis in control cells or cells treated with lat B (5 μM) to induce loss of F-actin (see also Movie S4). To confirm F-actin loss, actin filaments were visualized in fixed control and lat-B-treated cells using fluorophore-labeled phalloidin. Scale bars represent 20 μm.
(C) Plot shows NEB to anaphase timing for control cells (n = 146), lat-B-treated cells (n = 153), and cells treated with a low dose (7 ng/ml) of nocodazole (n = 128). Mitotic timing was similar in control and lat-B-treated cells (p = 0.09, unpaired t test), whereas nocodazole delayed anaphase onset (p < , unpaired t test).
(D) Actin is required for spindle assembly in cells growing under a soft gel. Schematic diagram showing the principle of the experiment. Cells were plated on a glass slide and then grown in the presence (right column) or absence (left column) of a covering gel and treated with cytochalasin D (0.5 μM; bottom row) or left untreated (top row).
(E) Mitotic phenotypes of HeLa cells in the four conditions described in (D) (n = 306, 679; 335, 503; in more than three experiments). Errors bars represent SD from the mean. Normal bipolar mitosis was decreased and multipolar spindles were increased in cells with a perturbed actin cortex growing under gels (p < 0.01, p < 0.05, unpaired t test, respectively).
See also Figure S6.
Developmental Cell  , DOI: ( /j.devcel )
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8. Figure 7 A Model for the Role of Cell Geometry in Mitosis
Schematic diagram summarizing the consequences of mitotic cell rounding defects for mitotic spindle formation.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions